Inhibiting ataxia telangiectasia and rad3-related protein (atr)

ABSTRACT

Novel compounds inhibiting ATR protein kinase include compounds of formula (I) disclosed herein, as well as liposome formulations comprising ATR protein kinase inhibitor compounds. The compositions are useful for the treatment of cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/069,092 filed on Jul. 10, 2018, which application is a 35 U.S.C. §371 filing of International Application No. PCT/US2017/012939, filedJan. 11, 2017, which application claims priority to U.S. ProvisionalPatent Application No. 62/277,262, filed Jan. 11, 2016, U.S. ProvisionalPatent Application No. 62/420,258, filed Nov. 10, 2016, and U.S.Provisional Patent Application No. 62/444,172, filed Jan. 9, 2017, eachof which are incorporated by reference into the present application intheir entirety and for all purposes.

FIELD

This disclosure relates to compounds and related methods of inhibitingataxia telangiectasia and Rad3-related protein (ATR), including methodsand compounds useful for the treatment of cancer.

BACKGROUND

The ataxia-telangiectasia and Rad3-related (ATR) kinase is aserine/threonine protein kinase believed to be involved in the cellularDNA damage repair processes and cell cycle signaling. ATR kinase actswith ATM (“ataxia telangiectasia mutated”) kinase and other proteins toregulate a cell's response to DNA damage, commonly referred to as theDNA Damage Response (“DDR”). The DDR is believed to stimulate DNArepair, promote survival and stalls cell cycle progression by activatingcell cycle checkpoints, which provide time for repair. Without the DDR,cells are much more sensitive to DNA damage and readily die from DNAlesions induced by endogenous cellular processes such as DNA replicationor exogenous DNA damaging agents commonly used in cancer therapy.

The disruption of ATR function (e.g. by gene deletion) has been shown topromote cancer cell death both in the absence and presence of DNAdamaging agents. Mutations of ATR have been linked to cancers of thestomach and endometrium, and lead to increased sensitivity to ionizingradiation and abolished cell cycle checkpoints. ATR is essential for theviability of somatic cells, and deletion of ATR has been shown to resultin loss of damage checkpoint responses and cell death. See Cortez etal., Science 294: 1713-1716 (2001). ATR is also essential for thestability of fragile sites, and low ATR expression in Seckel syndromepatients results in increased chromosomal breakage following replicationstress. See Casper et al., Am. J. Hum. Genet 75: 654-660 (2004). Thereplication protein A (RPA) complex recruits ATR, and its interactingprotein ATRIP, to sites of DNA damage, and ATR itself mediates theactivation of the CHK1 signaling cascade. See Zou et al., Science300:1542-1548 (2003). ATR, like its related checkpoint kinase ATM,phosphorylates RAD17 early in a cascade that is critical to forcheckpoint signaling in DNA-damaged cells. See Bao et al., Nature 411:969-974 (2001). It is believed that ATR is particularly essential in theearly mammalian embryo, to sense incomplete DNA replication and preventmitotic catastrophe.

However, while DNA-damaging chemotherapy agents and ionizing radiation(IR) therapy have provided initial therapeutic benefits to cancerpatients, existing therapies have lost clinical efficacy (e.g., due totumor cell DNA repair responses). In vivo effects of an ATR inhibitorand a DNA damaging agent have shown some promise in the selectivetreatment of cancer compared to normal cells, particularly in treatingtumor cells deficient in the G1 check point control (which may dependmore on the ATR for survival).

There remains a need for the development of potent and selectivetherapies to deliver ATR inhibitors for the treatment of cancer, eitheras single agents or as part of combination therapies (e.g., incombination with chemotherapy and/or radiation therapy).

SUMMARY

Applicants have discovered novel chemical compounds useful forinhibiting ataxia-telangiectasia and Rad3-related (ATR) kinase and thetreatment of cancer, and liposome formulations of certain inhibitors ofATR protein kinase having desirable properties (e.g., extended half-lifein blood circulation and efficacy in treating tumors). The inventionsare based in part on the discovery of certain novel compounds forinhibiting ATR protein kinase, as well as extended plasma half-lives andenhanced antitumor efficacy of certain liposomal formulations of ATRprotein kinase inhibitor compounds.

In a first embodiment, novel compounds of formula (I) are useful forinhibiting ataxia-telangiectasia and Rad3-related (ATR) kinase and thetreatment of cancer:

or a pharmaceutically acceptable salt thereof, wherein R is a moietycomprising an amine with a pK_(a) of greater than 7.0 (preferablygreater than 8.0, and most preferably at least about 9.5). The compoundsof formula (I) preferably include one or more tertiary amine moieties atR selected to provide desired inhibition of ATR and/or liposomeformation and stability characteristics. In some examples, R is aheterocyclic moiety comprising a first tertiary substituted nitrogen,preferably substituted with an alklyamino moiety comprising a secondtertiary substituted nitrogen. In particular, compounds of formula (I)include those where R can be a moiety of the formula:

wherein A¹ is either absent or is an alkyl (e.g., C₁-C₄ alkyl(preferably —(CH₂)₂—), and R¹ is lower (e.g., C₁-C₄) alkylamino. In oneembodiment, R¹ is (C₁-C₄ alkyl)-NR^(a)R^(b), wherein R^(a) and R^(b) areeach independently C₁-C₄ alkyl, e.g., R¹ is —(CH₂)₂—N(CH₃)(CH₃). Inanother embodiment, R¹ is NR^(a)R^(b), wherein R^(a) and R^(b) are eachindependently C₁-C₄ alkyl, e.g., R¹ is —N(CH₂CH₃)(CH₂CH₃).

In another embodiment of formula (I), R is —N(H)(C₁-C₄alkyl)-NR^(a)R^(b), wherein R^(a) and R^(b) are each independently C₁-C₄alkyl, or R is —(G)-NR^(a)R^(b), wherein R^(a) and R^(b) are eachindependently C₁-C₄ alkyl, wherein G is C₁-C₄ alkyl, and wherein G canbe further substituted with C₁-C₄ alkyl.

In another embodiment of formula (I), R can be a moiety of the formula:

wherein R^(c) and R^(d) are each independently C₁-C₄ alkyl.

Preferred examples include liposomes comprising compounds selected fromthe group consisting of compounds 1, 2, 3, 4, 5, or 6:

In a second embodiment, liposome formulations of ATR inhibitor compoundscan include a compound of formula (I) or other ATR inhibitor compound(s)(e.g., comparative Compound A) encapsulated with a polyanion (e.g., apolyanionized sugar such as sucrose octasulfate, or a suitablepolyanionized polyol) in a unilamellar vesicle formed from one or moreliposome-forming lipids (e.g., hydrogenated soy phosphatidylcholine(HSPC)), cholesterol and a polymer-conjugated lipid (e.g.,methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glyceryl (PEG2000-DSG).The liposome-forming lipid preferably comprises one or morephospholipids, with the ratio of the phospholipid(s) and the cholesterolselected to provide a desired amount of liposome membrane rigidity whilemaintaining a sufficiently reduced amount of leakage of the compound offormula (I) from the liposome. The type and amount of polymer-conjugatedlipid can be selected to provide desirable levels of protein binding,liposome stability and circulation time in the blood stream. In someexamples, the liposome vesicle comprises HSPC and cholesterol in a 3:2molar ratio. In particular, the liposome can comprise a vesicleconsisting of HSPC, cholesterol and PEG2000-DSG in a 3:2:0.15 molarratio. The compound of formula (I) can be entrapped within the liposomewith a suitable polyanion, such as sucrose octasulfate. In someexamples, the liposome encapsulates the compound of formula (I) andsucrose octasulfate in a ratio at or near the stoichiometric ratio ofthe compound of formula (I) and the sucrose octasulfate.

One specific example provides a liposome having a vesicle formed fromHSPC, cholesterol and PEG2000-DSG in a 3:2:0.15 molar ratio,encapsulating sucrose octasulfate and Compound 5. Another exampleprovides a liposome having a vesicle formed from HSPC, cholesterol andPEG2000-DSG in a 3:2:0.15 molar ratio, encapsulating sucrose octasulfateand Compound 5.

Another specific example provides a liposome having a vesicle formedfrom HSPC, cholesterol and PEG2000-DSG in a 3:2:0.15 molar ratio,encapsulating sucrose octasulfate and Compound 6.

Another specific example provides a liposome having a vesicle formedfrom HSPC, cholesterol and PEG2000-DSG in a 3:2:0.15 molar ratio,encapsulating sucrose octasulfate and Compound A.

The ATR inhibitor compounds and/or liposome formulations thereofdisclosed herein can be used in therapy and methods of treatment. Insome embodiments, the therapy is treatment of cancer. When used as atherapy, the liposome composition may be used in a treatment regimenwith one or more other compounds or compositions (e.g., in combinationwith an irinotecan

liposome formulation such as MM-398). The administration of the liposomecomposition with one or more other compounds or compositions may besimultaneous, separate or sequential. The one or more other compounds orcompositions may be further therapeutics, e.g. further anticanceragents, or may be compounds which are designed to ameliorate thenegative side effects of the therapeutic agents.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a first chemical reaction scheme useful in manufacturingcertain compounds disclosed herein.

FIG. 2 is a second chemical reaction scheme useful in manufacturingcertain compounds disclosed herein.

FIG. 3 is a third chemical reaction scheme useful in manufacturingcertain compounds disclosed herein.

FIG. 4 is a fourth chemical reaction scheme useful in manufacturingcertain compounds disclosed herein.

FIG. 5 is a graph showing the blood pharmacokinetics of liposomal ATRinhibitors, as discussed in Example 8.

FIG. 6 is a graph showing the antitumor efficacy of liposomal Compound Ain combinations with MM-398 in cervical MS751 xenograft model, asdescribed in Example 9A.

FIG. 7 is a graph showing the antitumor efficacy of liposomal Compound Ain combinations with MM-398 in cervical C33A xenograft model, asdescribed in Example 9A.

FIG. 8 is a graph showing the antitumor efficacy of liposomal Compound Ain combinations with MM-398 in cervical C33A xenograft model, accordingto Example 9A.

FIG. 9 is a graph showing the tolerability of liposomal Compound A incombination with MM-398 in cervical MS751, according to Example 9A.

FIG. 10A and FIG. 10B are each a graph, showing the efficacy ofliposomal Compound 5 in combination with MM-398 in NCI-H2170 (FIG. 10A)or DMS-114 (FIG. 10B) mice xenograft models, as discussed in Example 10.

FIG. 11A and FIG. 11B are each a graph showing the Kaplan-Meyer survivalcurves representing efficacy of liposomal Compound 5 in combination withMM-398 in NCI-H2170 (FIG. 11A) and DMS-114 (FIG. 11B) a mouse xenograftmodel, as discussed in Example 10.

FIG. 12A and FIG. 12B are each a graph showing Tolerability of liposomalCompound 5 in combination with MM-398 in NCI-H2170 (FIG. 12A) or DMS-114(FIG. 12B) in a mouse xenograft model.

FIG. 13A and FIG. 13B are graphs showing the efficacy of liposomalCompound 5 in combination with MM-398 in Calu-6 (FIG. 13A) or COLO-699(FIG. 13B) mice xenograft models, as described in Example 11.

FIG. 14A is a graph illustrating the in vitro monotherapy cell kill ofCompound 6 and Compound 5 in a panel of lung cancer cell lines.

FIG. 14B is a graph illustrating the effect of Compound 5 and Compound 6in combination with three chemotherapeutic agents (Carboplatin,Gemcitabine, Compound B).

FIG. 15 is a graph showing the IC₅₀ shift values measured in Sum190PTcell line (Triple Negative Breast Cancer, TNBC) for the ATR proteinkinase inhibitor Compound 6 of Example 2, with and without variousconcentrations of Compound B.

FIG. 16A is a graph showing the IC₅₀ shift values of a combination ofATR protein kinase inhibitor Compound 6 of Example 2, measured inMDA-MB-453 TNBC cancer cell line.

FIG. 16B is a graph showing the IC₅₀ shift values of a combination ofATR protein kinase inhibitor Compound A, measured in MDA-MB-453 TNBCcancer cell line.

FIG. 17 is a western blot analysis of obtained from lung cancer cellsDMS-114 exposed either to Gemcitabine [16 nM] or ATR inhibitor (CompoundA or Compound 5 [1 uM]) alone or in combination in-vitro.

FIG. 18A-B is cell death and growth assay performed combining Compound Aand Gemcitabine at various concentrations in U2OS cells. The results arecompared back to a prediction of cell growth and death with thecombination of the two compounds (FIG. 18A). The number of living andapoptotic cells is also determined at set concentrations of Compound A(1 μM) and Gemcitabine (0.04 μM) in USO2 cells (FIG. 18B).

FIG. 19 shows Compound A being tested alone and in combination with SN38at set concentrations (1 μM and 0.2 μM, respectively) in several lungcancer cell lines (NCI-H520 and NCI-H596) and U2OS cells, with thenumber of cells monitored over time.

FIG. 20 is a graph illustrating the effect of Compound A and SN38 incombination and alone in the cervical cancer cell line MS751.

FIG. 21A-C demonstrates the effects of combining Compound A or Compound5 with Gemcitabine. FIG. 21A is a heat map illustrating the effect ofcombining Compound A or Compound 5 with Gemcitabine at variousconcentrations in U2OS, H358, and A549 cell lines. Microscopy images ofcells treated with set concentrations of Compound 5 and Gemcitabine orCompound A and Gemcitabine in cells are shown (FIG. 21B). Proliferationassays of USO2 and H358 cells with set concentrations are shown as well(FIG. 21C).

FIG. 22A-B illustrate growth curves of A549 cells with Compound A orCompound 5 in combination with SN38 (FIG. 22A) or alone (FIG. 22B).

FIG. 23 shows IC50 values (μM) in several cell lines using a setconcentration of Gemcitabine with varying concentrations of Compound Aor Compound 5.

FIG. 24 shows a summary of the lung cancer cell lines that areresponsive to Compound A or Compound 5 in combination with Gemcitabineor SN38.

FIG. 25A-B show various ATR inhibitors tested for their ability toinhibit ATR (on-target) and ATM (off-target). Inhibition is reported asIC50 in nM (FIG. 25A). Additional “off-target” kinases are tested withCompound A or Compound 5 as well (FIG. 25B).

FIG. 26A-F shows on-target results with Compound A or Compound 5 in A549lung cancer cells (FIG. 26A-B), H23 lung cancer cells (FIG. 26C-D), andDMS-114 cells (FIG. 26E-F). CHK1 S345 phosphorylation, a readout of ATRinhibition, is measured by western blot. Each compound is used with afixed concentration of Gemcitabine as well.

FIG. 27A-B shows further on-target analysis on the HCC-70 TNBC,MDA-MB-468 TNBC, and DMS-114 cell lines. A set concentration of SN38 isused with a range of Compound A or Compound 5 concentrations. Variouson-target activity parameters are tested and measured by western blot(FIG. 27A-B)

FIG. 28 shows cell cycle stage profiles of SUM149 cells 24 hours afteraddition of SN38 and Compound A or Compound 5.

FIG. 29 shows DMS-114 lung xenograft models used to measure the effectsof Ls-Compound A or Ls-Compound 5 in combination with MM398. A setdosage of MM398 is used (5 mpk) with two different dosages of Compound Aor Compound 5 (20 mpk or 80 mpk). The effects of treatment are assayedby measuring the levels of CHK1 S345 phosphorylation.

FIG. 30A-C illustrates the effects of Ls-Compound A with MM398 in theSUM-149 cell line. The effects of the treatment are assayed by measuringphosphorylated levels of RPA2 (FIG. 30A), DNAPK, CHK1, and γH2AX (FIG.30B). Ls-Compound 5 is also tested without the combination of MM398(FIG. 30C).

FIG. 31 is a graph showing the efficacy of liposomal Compound 5 incombination with MM-398 in SUM-149 mice xenograft model.

FIG. 32 is a graph showing the tolerability of liposomal Compound 5 incombination with MM-398 in SUM-149 mice xenograft models

FIG. 33 Basal levels of various PD markers associated with the DNAdamage response pathway, quantified by Western blot over a panel of celllines.

FIG. 34A schematic illustrating how the integral score is calculated foreach well in the dynamic cell viability assay.

FIG. 35 Correlation of basal MRE11 protein expression (quantified byWestern blot) and the integral score, a measure of dynamic cellviability, across lung cancer cell lines exposed to the ATR inhibitorCompound 5 and/or SN38.

FIG. 36 Correlation of basal ATM protein expression (quantified byWestern blot) and the integral score, a measure of dynamic cellviability, across lung cancer cell lines exposed to the ATR inhibitorCompound 5 and/or SN38.

FIG. 37 Correlation of basal NBS protein expression (quantified byWestern blot) and the integral score, a measure of dynamic cellviability, across lung cancer cell lines exposed to the ATR inhibitorCompound 5and/or SN38.

FIG. 38 Correlation of basal NBS protein expression (quantified byWestern blot) and the integral score, a measure of dynamic cellviability, across p53 functionally impaired lung cancer cell linesexposed to the ATR inhibitor Compound 5 and/or SN38.

FIG. 39 Correlation of basal NBS protein expression (quantified byWestern blot) and the integral score, a measure of dynamic cellviability, across p53 functionally impaired lung cancer cell linesexposed to the ATR inhibitor Compound 5 and/or SN38.

FIG. 40A-F Fold change in cancer cell line NCIH1299 pharmacodynamicsmarkers after exposure to ATR inhibition and/or SN38.

FIG. 41A-F Fold change in cancer cell line NCIH460 pharmacodynamicsmarkers after exposure to ATR inhibition and/or SN38.

FIG. 42A-F Fold change in cancer cell line DMS114 pharmacodynamicsmarkers after exposure to ATR inhibition and/or SN38.

FIG. 43A-F Fold change in cancer cell line HCC70 pharmacodynamicsmarkers after exposure to ATR inhibition and/or SN38.

FIG. 44A-F Fold change in cancer cell line MDAMB468 pharmacodynamicsmarkers after exposure to ATR inhibition and/or SN38.

FIG. 45 Western blots of pharmacodynamics markers in cancer cell lineA549 after 6 or 18 hours exposure to ATR inhibition and/or gemcitabine.

FIG. 46 Western blots of pharmacodynamics markers in cancer cell lineNCIH23 after 6 or 18 hours exposure to ATR inhibition and/orgemcitabine.

FIG. 47 Western blots of pharmacodynamics markers in cancer cell lineDMS114 after 6 or 18 hours exposure to ATR inhibition and/orgemcitabine.

FIG. 48 Western blots of pharmacodynamics markers in cancer cell lineU2OS after 6 or 18 hours exposure to ATR inhibition and/or gemcitabine.

FIG. 49 Western blots of pharmacodynamics markers in cancer cell lineNCIH460 after 6 or 18 hours exposure to ATR inhibition and/orgemcitabine.

FIG. 50 Western blots of pharmacodynamics markers in cancer cell lineHCC827 after 6 or 18 hours exposure to ATR inhibition and/orgemcitabine.

FIG. 51 Western blots of pharmacodynamics markers in a panel ofcolorectal cancer cell lines after 18 hours exposure to Compound 5and/or SN38.

FIG. 52 Normalized quantification of phosphorylated Chk1 levels in apanel of colorectal cancer cell lines after 18 hours exposure toCompound 5 and/or SN38 (the signal for each cell line is normalized tothe signal in the presence of SN38 alone).

FIG. 53 Normalized quantification of phosphorylated RPA2 levels in apanel of colorectal cancer cell lines after 18 hours exposure toCompound 5 and/or SN38 (the signal for each cell line is normalized tothe signal in the presence of SN38 alone).

FIG. 54 Normalized quantification of γH2AX levels in a panel ofcolorectal cancer cell lines after 18 hours exposure to Compound 5and/or SN38 (the signal for each cell line is normalized to the signalin the presence of SN38 alone).

DETAILED DESCRIPTION

Novel compounds for inhibiting ATR protein kinase are described byformula (I):

or a pharmaceutically acceptable salt thereof, wherein R is a moietycomprising an amine with a pK_(a) of greater than 7.0 (preferablygreater than 8.0, and most preferably at least about 9.5), selected toprovide a plasma half-life of at least about 5 hours in mice (obtainedaccording to Example 7). Preferably, R includes an amine-substitutedalkyl moiety with 4-12 carbons. R can be selected to include only acombination of tertiary-substituted amine and hydrogenated alkyl groups.R preferably further includes a tertiary-alkyl substituted amine havinga pK_(a) of at least 7, but most preferably at least about 9.5 (e.g., apK_(a) of about 9.5-10.5). Examples of compounds of formula (I), orpharmaceutically acceptable salts thereof, include Compounds 1-6 (seeExamples 1-6):

The compounds of formula (I) preferably include one or more tertiaryamine moieties at R selected to provide desired inhibition of ATR and/orliposome formation and stability characteristics. In some examples, R isa heterocyclic moiety comprising a first tertiary substituted nitrogen,preferably substituted with an alklyamino moiety comprising a secondtertiary substituted nitrogen. In particular, compounds of formula (I)include those where R can be a moiety of the formula:

wherein A¹ is either absent or is an alkyl (e.g., C₁-C₄ alkyl(preferably —(CH₂)₂—), and R¹ is lower (e.g., C₁-C₄) alkylamino. In oneembodiment, R¹ is (C₁-C₄ alkyl)-NR^(a)R^(b), wherein R^(a) and R^(b) areeach independently C₁-C₄ alkyl, e.g., R¹ is —(CH₂)₂—N(CH₃)(CH₃). Inanother embodiment, R¹ is NR^(a)R^(b), wherein R^(a) and R^(b) are eachindependently C₁-C₄ alkyl, e.g., R¹ is —N(CH₂CH₃)(CH₂CH₃).

In another embodiment of formula (I), R is —N(H)(C₁-C₄alkyl)-NR^(a)R^(b), wherein R^(a) and R^(b) are each independently C₁-C₄alkyl, or R is -(G)-NR^(a)R^(b), wherein R^(a) and R^(b) are eachindependently C₁-C₄ alkyl, wherein G is C₁-C₄ alkyl, and wherein G canbe further substituted with C₁-C₄ alkyl.

In another embodiment of formula (I), R can be a moiety of the formula:

wherein R^(c) and R^(d) are each independently C₁-C₄ alkyl.

Preferred examples include liposomes comprising compounds selected fromthe group consisting of compounds 1, 2, 3, 4, 5, or 6 above. In someexamples, the compound is compound 5 or compound 6.

The compound of formula (I) can have the chemical structure of formula(Ia), or a pharmaceutically acceptable salt thereof, wherein R′ is atertiary alkyl substituted amine having a pK_(a) of about 9.5 orgreater:

Examples of compounds of formula (Ia) include compound 5 disclosedherein (e.g., Examples 1). In one embodiment of formula (Ia), R′ isNR^(a)R^(b), wherein R^(a) and R^(b) are each independently C₁-C₄ alkyl.

Liposome formulations of ATR protein kinase inhibitor compounds (e.g.,as described in Example 7) can provide desirable pharmacokineticproperties such as enhanced plasma half-life of 5 hours or more in themouse model described in Example 8. The liposomes typically comprisevesicles containing one or more lipid bilayers enclosing an aqueousinterior. Liposome compositions usually include liposomes in a medium,such as an aqueous fluid exterior to the liposome. Liposome lipids caninclude amphiphilic lipid components that, upon contact with aqueousmedium, spontaneously form bilayer membranes, such as phospholipids, forexample, phosphatidylcholines. Liposomes also can includemembrane-rigidifying components, such as sterols, for example,cholesterol. In some cases, liposomes also include lipids conjugated tohydrophilic polymers, such as, polyethyleneglycol (PEG) lipidderivatives that may reduce the tendency of liposomes to aggregate andalso have other beneficial effects.

The liposome formulation can include a compound of formula (I)encapsulated with a polyanion (e.g., a polyanionized sugar such assucrose octasulfate, or a suitable polyanionized polyol) in aunilamellar vesicle formed from one or more liposome-forming lipids(e.g., hydrogenated soy phosphatidylcholine (HSPC)), cholesterol and apolymer-conjugated lipid (e.g., methoxy-poly(ethyleneglycol)-1,2-distearoyl-sn-glyceryl (PEG2000-DSG). The liposome-forminglipid preferably comprises one or more phospholipids, with the ratio ofthe phospholipid(s) and the cholesterol selected to provide a desiredamount of liposome membrane rigidity while maintaining a sufficientlyreduced amount of leakage of the compound of formula (I) from theliposome.

Liposomes typically have the size in a micron or submicron range and arewell recognized for their capacity to carry pharmaceutical substances,including anticancer drugs, such as irinotecan, and to change theirpharmaceutical properties in various beneficial ways. Methods ofpreparing and characterizing pharmaceutical liposome compositions areknown in the field (see, e.g., Lasic D. Liposomes: From physics toapplications, Elsevier, Amsterdam 1993; G. Greroriadis (ed.), LiposomeTechnology, 3^(rd) edition, vol. 1-3, CRC Press, Boca Raton, 2006; Honget al., U.S. Pat. No. 8,147,867, incorporated by reference herein intheir entirety for all purposes).

In some examples (e.g., Example 7), ATR protein kinase inhibitorcompositions can include a liposome comprising a ATR protein kinaseinhibitor compound encapsulated in a liposome with polyanion such as apolysulfated sugar (e.g., sucrose octasulfate). Sucrosofate, a fullysubstituted sulfate ester of sucrose having, in its fully protonatedform, the following structure:

Sucrosofate is also referred to as sucrose octasulfate orsucrooctasulfate (SOS). Methods of preparing sucrosofate in the form ofvarious salts, e.g., ammonium, sodium, or potassium salts, are wellknown in the field (e.g., U.S. Pat. No. 4,990,610, incorporated byreference herein in its entirety).

The ATR protein kinase inhibitor liposomes can be prepared in multiplesteps comprising the formation of a TEA containing liposome, followed byloading of an ATR protein kinase inhibitor compound (e.g., Compound A ora compound of formula (I)) into the liposome as the TEA leaves theliposome. For example, the ATR protein kinase inhibitor liposomes can beprepared by a process that includes the steps of (a) preparing aliposome containing triethylamine (TEA) as a triethylammonium salt ofsucrosofate (TEA-SOS), and (b) subsequently contacting the TEA-SOSliposome with irinotecan under conditions effective for the irinotecanto enter the liposome and to permit a corresponding amount of TEA toleave the liposome (thereby exhausting or reducing the concentrationgradient of TEA across the resulting liposome).

The first step can include forming the TEA-sucrosofate containingliposome by hydrating and dispersing the liposome lipids in the solutionof TEA sucrosofate. This can be performed, for example, by dissolvingthe lipids, including HSPC and cholesterol, in heated ethanol, anddispersing the dissolved and heated lipid solution in theTEA-sucrosofate aqueous solution at the temperature above the transitiontemperature (Tm) of the liposome lipid, e.g., 60° C. or greater. Thelipid dispersion can be formed into liposomes having the average size of75-125 nm (such as 80-120 nm, or in some embodiments, 90-115 nm), byextrusion through track-etched polycarbonate membranes with the definedpore size, e.g., 100 nm. The TEA-sucrosofate can include at least 8molar equivalents of TEA to each molar equivalent of sucrosofate toobtain a solution that can have a concentration of about 0.40-0.50 N,and a pH (e.g., about 6.5) that is selected to prevent unacceptabledegradation of the liposome phospholipid during the dispersion andextrusion steps (e.g., a pH selected to minimize the degradation of theliposome phospholipid during these steps). Then, the non-entrappedTEA-SOS can be removed from the liposome dispersion, e.g., by dialysis,gel chromatography, ion exchange or ultrafiltration prior to the drugencapsulation. The resulting liposomes can contain ATR protein kinaseinhibitor sucrosofate. These ATR inhibitor liposomes can be stabilizedby loading enough drug into the liposomes to reduce the amount of TEA inthe resulting liposome composition to a level that results in less thana given maximum level of lyso-PC formation after 180 days at 4° C., orless than a given maximum level of lyso-PC accumulation rate in theliposome composition during storage in a refrigerator at about 4° C.,or, more commonly, at 5±3° C., measured, e.g., in mg/mL/month, or % PCconversion into a lyso-PC over a unit time, such as, mol %lyso-PC/month. Next, the TEA exchanged from the liposomes into theexternal medium during the loading process, along with any unentrappedATR inhibitor, is typically removed from the liposomes by any suitableknown process(es) (e.g., by gel chromatography, dialysis, diafiltration,ion exchange or ultrafiltration). The liposome external medium can beexchanged for an injectable isotonic fluid (e.g. isotonic solution ofsodium chloride), buffered at a desired pH.

The antitumor efficacy of various liposome formulations comprisingliposome encapsulated ATR protein kinase inhibitor compounds was testedin human cervical cancer cell lines (e.g., MS751, C33A and SiHa celllines, as shown in Example 9), and various lung cancer cell linesincluding lung squamous cell carcinoma cell line (e.g., NCI-H2170 cellline in Example 10), small cell lung carcinoma cell line (e.g., DMS-114cell line in Example 10), and human Calu-6 and COLO-699 cell lines(Example 11).

Referring to FIGS. 6-9 and Example 9, a liposome formulation of the ATRinhibitor Compound A (Example 7) was tested against three human cervicalcancer cell lines in mouse xenograph model (Example 9A), alone and incombination with the irinotecan liposome formulation MM398 (Example 9B).Greater tumor volume was observed over time for the liposomal Compound Aformulation of Example 7 compared to the control experiment for 2 of the3 cervical cancer cell lines (MS571 and C33A). However, administeringthe irinotecan liposome MM398 (Example 9B) in combination with theCompound A liposome formulation (Example 7) resulted in greatersuppression of tumor volume in all three cervical cancer cell lines thaneither administration of MM398 alone or liposomal Compound A alone.

Referring to FIGS. 10A-10B and FIGS. 11A-11B, a liposome formulation ofthe ATR inhibitor Compound 5 of formula (I) and formula (Ia) (thecompound of Example 1 formulated as a liposome as described in Example7) was tested against two lung cancer cell lines in a mouse xenographmodel (Example 10), alone and in combination with the irinotecanliposome formulation MM398 (Example 9B). Referring to FIG. 10A and FIG.10B, the administration of the liposome formulation of Compound 5reduced tumor volume in each cell line tested compared to the controlexperiment in Example 10, the combination of MM398 and the Compound 5liposome composition of Example 7 reduced the tumor volume in the mousemodel to a greater degree than either compound administeredindependently of the other. Similarly, the Kaplan-Meyer survival curvespresented in Example 10 (FIG. 11A and FIG. 11B) demonstrate increasedsurvival in mouse lung cancer xenograph testing when a combination ofboth the irinotecan liposome MM398 of Example 9B was administered incombination with the Compound 5 liposome formulation of Example 7 usingtwo different cell lines.

Referring to FIG. 12A and FIG. 12B, the tolerability of various liposomeformulations of ATR protein kinase inihibitor compounds was assessed inExample 10. Referring to FIG. 12A, the decline in mouse bodyweighttested in the NCI-H2170 mouse xenograph model was lowest over time forthe liposome formulation of Compound 5 (Example 7), compared to theirinotecan liposome MM398 (Example 9B), the control or the combinationof the liposome formulation of Compound 5 in combination with MM398.Referring to FIG. 12B, the decline in mouse bodyweight tested in theDMS-114 mouse xenograph model was lowest over time for the thecombination of the liposome formulation of Compound 5 in combinationwith MM398, compared to the liposome formulation of Compound 5 (Example7), or the irinotecan liposome MM398 (Example 9B) administeredindependently.

Referring to FIG. 13A and FIG. 13B, administering a combination of theirinotecan liposome MM398 (Example 9B) with the liposome formulation ofATR protein kinase inhibitor Compound 5 resulted in the greatestreduction in tumor volume in both the Calu-6 and COLO699 cell lines inmice xenograft models, compared to the control, the administration ofMM398 irinotecan liposome alone, administration of the Compound Aliposome formulation (Example 7) or the combination of the MM398irinotecan liposome (Example 9B) with the Compound A liposomeformulation (Example 7).

EXAMPLES

The following examples illustrate some embodiments of the invention. Theexamples and preparations which follow are provided to enable thoseskilled in the art to more clearly understand and to practice these andother embodiments present invention. They should not be considered aslimiting the scope of the invention, but merely as being illustrativeand representative thereof.

ATR peptide can be expressed and isolated using a variety of methodsknown in the literature (see e.g., Ünsal-Kaçmaz et al, PNAS 99: 10, pp6673-6678, May 14, 2002; see also Kumagai et al. Cell 124, pp 943-955,Mar. 10, 2006; Unsal-Kacmaz et al. Molecular and Cellular Biology,February 2004, p 1292-1300; and Hall-Jackson et al. Oncogene 1999, 18,6707-6713).

Compound A can be obtained by methods disclosed (for example) inpublication WO2010/071827A1 (published Jun. 24, 2010), portions of whichrelating to the synthesis and use of compound II-A-7 are incorporatedherein by reference. The structure of Compound A is as follows:

Various compounds of formula (I) can be prepared as described herein,and summarized in the table below.

TABLE 1 Selected Compounds of Formula (I) (I)

Compound R Example Compound 5

1 Compound 6

2 Compound 2

3 Compound 4

4 Compound 3

5 Compound 1

6

The examples 1, 2, 3 and 6 were prepared in a one-pot Suzukicross-coupling using boronic ester generated in-situ shown in Scheme 1in FIG. 1 . Referring to FIG. 1 , the synthesis of Intermediate 3:1-bromo-4-(2-bromoethylsulfonyl)benzene can be obtained as describedbelow.

To a solution of Intermediate 2 (35 g, 133 mmol) in DCM (400 mL) wasadded PBr₃ (40 g, 146 mmol) at 0° C. dropwise. Then the mixture wasstirred at rt overnight. Water (15 mL) was added to quench the reaction.Then the resultant was washed with water (120 mL) and brine (120 mL).The organic phase was concentrated to afford 20 g of crude 3 as yellowoil, which was used in the next step without further purification.

Referring to FIG. 1 , the synthesis of intermediate 2 can be performedas described below:

To a solution of Intermediate 1 (45 g, 194 mmol) in DCM (500 ml) wasadded m-CPBA (134 g, 776 mmol) at rt in several batches. Then themixture was stirred at rt overnight. The reaction mixture was filteredand DCM (500 ml) was added to wash the solid. The filtrate was washedwith NaOH (1M, 300 mL×3) and brine (300 mL). The organic layer wasconcentrated to dryness to afford 36 g of 2 (70%) as white solid.

Referring to FIG. 1 , the synthesis of intermediate 1 can be performedas described below.

To a solution of 4-bromobenzenethiol (45 g, 238 mmol) in MeCN (600 ml)was added K₂CO₃ (60 g, 476 mmol) and NaI (36 g, 238 mmol). The mixturewas stirred at rt for 10 minutes. Then 2-bromoethanol was addeddropwise. After addition, the mixture was stirred at rt overnight. Thereaction mixture was filtered and the filtrate was concentrated todryness. The residue was purified by silica-gel column to afford 45 g of1 (81%) as light yellow oil.

Block B can be prepared according to scheme 2 in FIG. 2 . Referring toFIG. 2 , the synthesis of Block B can be performed as described below.

To a solution of Intermediate 6 (6.0 g, 27.4 mmol) in DMSO (30 mL) wasadded CDI (8.9 g, 54.8 mmol), DIPEA (3.8 g, 30.1 mmol) and DMAP (0.17 g,1.37 mmol). The solution was stirred at rt for 4 hours. Aniline (2.5 g,27.4 mmol) was added and the mixture was stirred at rt overnight. Waterwas added and the formed solid was collected by filtration. The crudeproduct was purified by silica-gel column to afford 2.5 g of Block B(31%) as yellow solid.

LC-MS (M+1): 293.2; ¹H NMR (400 MHz, DMSO-d₆) δ 10.28 (s, 1H), 8.42 (s,1H), 7.78 (d, J=8.0 Hz, 2H), 7.74 (s, 2H), 7.36 (t, J=8.0 Hz, 2H), 7.13(t, J=7.6 Hz, 1H).

Referring again to FIG. 2 , the synthesis of Intermediate 6 can beperformed as described below.

To the solution of methyl 3-amino-6-bromopyrazine-2-carboxylate (10.0 g43.1 mmol) in MeOH (70 mL) was added a solution of LiOH (9.0 g, 215mmol) in water (70 mL). The mixture was stirred at 90° C. for 3 hours.The reaction mixture was cooled to rt and acidified to PH=4˜5 with HCl(2 M). The mixture was filtered to afford 7.4 g of 6 (79%) as yellowsolid.

LC-MS (M+1): 218.0; 1H NMR (400 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.59 (br,2H).

Example 1: Synthesis of Compound 5(3-amino-6-(4-((2-(4-(2-(dimethylamino)ethyl)piperidin-1-yl)ethyl)sulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide)

Exact Mass: 536.26; Molecular Weight: 536.70; Compound 5; More basic;143 mg; Yield 8.2%; pK_(a) 10.00.

To a solution of2-(1-(2-((4-bromophenyl)sulfonypethyl)piperidin-4-yl)-N,N-dimethylethan-1-amine(Block A1) (261 mg, 0.648 mmol) in anhydrous dioxane (3 ml) was addedpotassium acetate (191 mg, 1.944 mmol) and Bis(pinacolato) diborane (246mg, 0.971 mmol), the reaction vessel was degassed by repeatingvacuum/nitrogen cycle and then was added Pd(dppf)₂Cl₂. CH₂Cl₂ (53 mg,0.0648 mmol), again was degassed and the reaction was heated at 90° C.for 2 hours under nitrogen. The reaction was then cooled down to roomtemperature and was added 3-amino-6-bromo-N-phenylpyrazine-2-carboxamide(Block B), 2M K₂CO₃ (1 ml) and degassed and purged with nitrogen. ThePd(PPh₃)₄ (75 mg, 0.0648 mmol) was added. The reaction was heated at100° C. for 4 hrs. The reaction was cooled to room temperature anddiluted with ethyl acetate and washed with brine three times and organiclayer was dried over Na₂SO₄. Upon removal of solvent on the rotovap, adark oil residual crude product was obtained and it was purified onsilica-gel column chromatography (Reveleris Flash Chromatography System)using 0-15% methanol in dichloromethane as an eluent. Desired productwas obtained as a yellow solid (149 mg, yield 43%). MS (M+H)+ 537; ¹HNMR (400 MHz, DMSO-d₆): δ 10.45 (s, 1H), 9.03 (s, 1H), 8.49 (d, 2H, 6.8Hz), 7.95 (d, 2H, 6.8 Hz), 7.88 (s, br, 2H), 7.81 (d, 2H, 8.8 Hz), 7.40(t, 2H, 7.2 Hz), 7.18 (t, 1H, 7.2 Hz), 3.53 (t, 2H, 7.2 Hz), 2.64 (d,2H, 11.6 Hz), 2.55 (t, 2H, 7.2 Hz), 2.05 (m, 2H), 1.98 (s, 6H), 1.17 (m,2H), 1.42 (d, 2H, 12.0 Hz), 1.18 (m, 3H), 0.78 (m, 2H).

High resolution mass (Thermo Scientific™ Q Exactive™ hybridquadrupole-Orbitrap mass spectrometer): Calculated forC₂₈H₃₆N₆O₃S+Proton (1.00728)=537.2642; theoretical m/z of single chargedion: 537.2642; Found: 537.2636.

Example 2: Synthesis of Compound 6(3-amino-6-(4-((2-(4-(diethylamino)piperidin-1yl)ethyl)sulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide)

Exact Mass: 536.26; Molecular Weight: 536.70; Compound 6; More basic; 53mg; Yield 11.1%; pK_(a) 9.81.

The example 2 was prepared in a similar fashion using Block A2(1-(2-((4-bromophenyl)sulfonyl)ethyl)-N,N-diethylpiperidin-4-amine), ayellow solid was obtained (52 mg, yield 24%). MS (M+H)+ 537; ¹H NMR (400MHz, DMSO-d₆): δ 10.45 (s, 1H), 9.05 (s, 1H), 8.51 (d, 2H, 8.4 Hz), 7.94(d, 2H, 8.8 Hz), 7.87 (s, br, 2H), 7.79 (d, 2H, 8.8 Hz), 7.42 (t, 2H,8.4 Hz), 7.18(t, 1H, 7.2 Hz), 3.54 (t, 2H, 6.4 Hz), 2.65 (d, 2H, 11.2Hz), 2.56 (t, 2H, 6.4 Hz), 2.20 (q, 4H, 6.8 Hz), 1.71 (t, 2, 10.4 Hz),1.33 (d, 2H, 12.4 Hz), 0.85 (qd, 2H, 12.4 Hz), 0.74 (t, 6H, 7.2 Hz).

1-(2-((4-bromophenyl)sulfonyl)ethyl)-N,N-diethylpiperidin-4-amine (BlockA2) was prepared in a similar fashion using corresponding4-diethylaminopiperidine. A colorless oil was obtained (661 mg, 47%yield), MS (M+H)+ 403, 405.

Example 3: Synthesis of Compound 2(3-amino-6-(4-((2-(diethylamino)ethyl)sulfonyl)phenyl)-Nphenylpyrazine-2-carboxamide)

Exact Mass: 453.18; Molecular Weight: 453.56; Compound 2; Less basic; 98mg; Yield 7.7%; pK_(a) 7.46.

The compound of example 3 was prepared in a similar fashion usingintermediate Block A3,2-((4-bromophenyl)sulfonyl)-N,N-diethylethan-1-amine, a yellow solid wasobtained (98 mg, yield 11%). MS (M+H)+ 454; ¹H NMR (400 MHz, DMSO-d₆): δ10.46 (s, 1H), 9.05 (s, 1H), 8.51 (d, 2H, 6.8 Hz), 7.98 (d, 2H, 6.8 Hz),7.88 (s, br, 2H), 7.81 (d, 2H, 8.8 Hz), 7.41 (t, 2H, 7.2 Hz), 7.17 (t,1H, 7.2 Hz), 3.48 (dd, 2H, 6.8 Hz), 2.73 (m, 2H), 2.33 (q, 4H, 6.8 Hz),0.81 (t, 6H, 6.8 Hz).

Block A3 (2-((4-bromophenyl)sulfonyl)-N,N-diethylethan-1-amine) wasprepared in a similar fashion using corresponding 4-diethylamine. Acolorless oil was obtained (1.42 g, 73% yield), MS (M+H)+ 320, 322

Example 4: Synthesis of Compound 4(3-amino-6-(4-(((2-(dimethylamino)ethyl)-λ²-azanyl)sulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide)

Exact Mass: 439.16; Molecular Weight: 439.51; Compound 4; pK_(a) 8.36.

Compound 4 can be prepared as shown in scheme 3 in FIG. 3 . To a mixtureof Block B (150 mg, 0.51 mmol), Block F (153 mg, 0.56 mmol) and Na₂CO₃(216 mg, 2.0 mmol) in toluene/ethanol/water (2 mL/2 mL/2 mL) was addedPd(dppf)Cl₂ (30 mg). The mixture was stirred at 75° C. under argonatmosphere for 4 hours. The reaction mixture was concentrated todryness. The residue was purified by prep-HPLC to afford 100 mg of TM4(45%) as white solid.

LC-MS (M+1): 441.4; ¹H NMR (400 MHz, CD₃OD) δ 8.88 (s, 1H), 8.33 (dd,J=6.8 Hz, 1.6 Hz, 2H), 8.00 (dd, J=6.8 Hz, 1.6 Hz, 2H), 7.80 (dd, J=8.4Hz, 1.2 Hz, 2H), 7.41 (t, J=7.6 Hz, 2H), 7.19 (t, J=7.6 Hz, 1H), 3.10(t, J=6.4 Hz, 2H), 2.73 (t, J=6.4 Hz, 2H), 2.46 (s, 6H).

Referring again to Scheme 3 in FIG. 3 , the synthesis of Block F can beperformed as described below.

To a solution of Intermediate 7 (10.0 g, 32.6 mmol) in THF (200 mL) at−78° C. under argon atmosphere was added B(i-Pr)₃ (30.6 g, 163 mmol).Then n-BuLi (2.5 M, 65 mL) was added dropwise. The mixture was stirredat −78° C. for 2 hours and at then rt for another 16 hours. Water wasadded to quench the reaction. The mixture was concentrated to dryness.The residue was purified by prep-HPLC to afford 5.2 g of Block F (59%)as white solid.

LC-MS (M+1): 273.4; ¹H NMR (400 MHz, DMSO-d₆) δ 7.90-7.45 (m, 4H), 2.91(s, 2H), 2.69 (t, J=6.4 Hz, 2H), 2.18 (t, J=6.8 Hz, 2H), 2.03 (s, 6H).

Referring again to Scheme 3 in FIG. 3 , the synthesis of Intermediate 7can be performed as described below.

To a solution of 4-bromobenzene-1-sulfonyl chloride (20 g, 78.3 mmol) inDCM (300 mL) at 0° C. was added TEA (22 mL, 158 mmol), followed byN,N′-dimethylethane-1,2-diamine (8.3 g, 94.0 mmol). The resultingsolution was stirred at rt for 1 hour and then diluted with DCM (300mL). The solution was washed with water (200 mL) and brine (200 mL). Theorganic layer was concentrated to dryness. The residue was purified bysilica-gel column to afford 17.0 g of 7 (71%) as off-white solid.

Example 5: Synthesis of Compound 3(3-amino-6-(4-((2-(4-methylpiperazin-1-yl)ethyl)sulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide)

Exact Mass: 480.19; Molecular Weight: 480.59; Compound 3; pK_(a) 7.73.

The compound of Example 5 can be obtained by Scheme 4 shown in FIG. 4 .Intermediate 5 in Scheme 4 can be obtained as described below.

To a solution of Intermediate 4 (1.7 g, 5.0 mmol) in THF (30 mL) at −78° C. under argon atmosphere was added B(i-Pr)₃ (4.7 g, 25 mmol). Thenn-BuLi (2.5 M, 10 mL) was added dropwise. The mixture was stirred at−78° C. for 2 hours and at then rt for another 16 hours. Water was addedto quench the reaction. The mixture was concentrated to dryness. Theresidue was purified by prep-HPLC to afford 300 mg of 5 (19%) as whitesolid.

Referring again to FIG. 4 , Intermediate 4 in Scheme 4 can be obtainedas described below.

To a solution of Intermediate 3 (20 g, 60 mmol) in MeCN (300 mL) wasadded 1-methylpiperazine (9.0 g, 90 mmol) and K₂CO₃ (16.6 g, 120 mmol).The mixture was stirred at rt overnight. The reaction mixture wasfiltered and the filtrate was concentrated to dryness. The residue waspurified by silica-gel column to afford 15 g of 4 (71%) as pale solid.

Example 6: Synthesis of Compound 1(3-amino-6-(4-((1-(dimethylamino)propan-2-yl)sulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide)

Exact Mass: 439.17; Molecular Weight: 439.53; Compound 1; Less basic;427 mg; Yield 12.9%; pK_(a) 7.04.

The compound of Example 6 was prepared according to the procedure forpreparing Compound 1 given in J. Med. Chem. 2011, 54, 2320(supplementary materials) except using1-bromo-4-(2-bromoethylsulfonyl)benzene. A yellow solid was obtained(427 mg, yield 11%). MS (M+H)+ 440; ¹H NMR (400 MHz, DMSO-d₆): δ 10.46(s, 1H), 9.05 (s, 1H), 8.52 (d, 2H, 6.8 Hz), 7.93 (d, 2H, 6.8 Hz), 7.89(s, br, 2 H), 7.82 (d, 2H, 8.0 Hz), 7.40 (t, 2H, 7.2 Hz), 7.18 (t, 1H,7.2 Hz), 3.53 (t, 2H, 7.2 Hz), 2.64 (d, 2H, 11.6 Hz), 2.55 (t, 2H, 7.2Hz), 2.05 (m, 2H), 1.98 (s, 6H), 1.17 (m, 2H), 1.42 (d, 2H, 12.0 Hz),1.18 (m, 3H), 0.78 (m, 2H).

Example 7: Preparation of Liposomes with Entrapped Triethylammonium SOSSalts and Loading of ATRi into the Liposome

Sodium sucrose octasulfate (equivalent weight 144.8) is a sodium salt ofsucrose derivate in which all hydroxyl groups have formed sulfuric acidesters. Sixty gram of sucrose octasulfate (SOS) sodium salt weredissolved in in 150 ml of di water, heated with shaking (swirling) of a50° C. water bath. The solution was passed through a column packed withsulfonated polystyrene divinylbenzene copolymer cation exchange resinbeads (Dowex mesh, Dow Chemical Co.). The column was pre-equilibratedwith aqueous 3-3.6 M HCl to bring the resin in the hydrogen form andwashed with deionized water until the outflow shows conductivity of <1μS/cm. The eluent was monitored using a conductivity detector. The SOSfraction was collected corresponding to conductivity peak andimmediately titrated with neat triethylamine (TEA) solution to pH 6-6.5.The solution was analyzed for residual sodium by potentiometry using asodium-sensitive electrode and for SOS concentration using arefractometer. The solution having residual sodium less than 0.25% wasdiluted with di water to final SOS concentration 1.1 M and thensterile-filtered using Millipore 0.22 μm Steri-Top filter.

Cholesterol (Chol) was purchased from Avanti Polar Lipids, Alabaster,Ala, USA, hydrogenated soy phosphocholine (HSPC) andmethoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glyceryl (PEG2000-DSG)were obtained from Lipoid GmbH, Ludwigshafen, Germany.

Chol, HSPC and PEG2000-DSG were co-dissolved in 100% ethanol (200-proof,Sigma cat#: 459828) at a molar ratio of 3:2:0.15 at 65° C. The solutionof TEA-SOS (10 times the volume of added ethanol) was mixed with thelipid solution at 60-65° C. and stirred at this temperature until ahomogeneous milky suspension of multilamellar vesicles was formed. Thissuspension was extruded 3 times through 5 stacked polycarbonatetrack-etched filters (Corning Nuclepore) with the pore size of 100 nmusing argon pressure extruder (Lipex Biomembranes) at 60-65° C., andresulting unilamellar liposomes were quickly chilled in ice and thenstored at 4-6° C. before use. Concentration of phospholipids wasmeasured by phosphate assay and particle diameter was recorded on aMalvern Nanosizer.

Prior to drug loading, the TEA-SOS gradient was created by removingexcess of un-trapped TEA-SOS using gel-chromatography (Sepharose CL-4B,Pharmacia). Osmolarity of liposomes was balanced using 50% dextrosesolution. Final dextrose concentration was 15%.

ATR inhibitors were dissolved in 15% dextrose solutions in di water bytitration with 1 M HCl and heating at 45° C. and then filtered with 0.2micron NALGENE 13 mm Syringe Filters. Drug concentration in the solutionwas detected by HPLC. A stock solution of ATR inhibitors containing 9-10mg/mL of drugs was added to the liposomes at a drug/lipid ratio of 800mg/mmol phospholipids and pH was adjusted to pH 6.5 with 1M Hepes bufferand 0.1 N NaOH.

The liposome-drug mixture was incubated with occasional agitation for 30minutes at 65° C. The incubation mixture was quickly cooled down andincubated for 10 minutes at 0° C., then allowed to attain ambienttemperature. Un-encapsulated drug was removed by gel chromatography onSephadex G-25 (Amersham Pharmacia) eluted with HBS-6.5 buffer (5 mM2-(4-(2-hydroxyethyl)-piperazino)-ethylsulfonic acid (HEPES), 144 mMNaCl, pH 6.5). Liposome fractions eluted in the void volume werecombined, sterilized by 0.2 micron filtration, and stored at 4-6° C.before use. The liposomes were characterized by lipid concentration,drug concentration, and particle size (Table 2). All ATR inhibitorsshowed good loading efficacy except Compound 1, which at drug to lipidration higher than 400 g/mol formed aggregates and participated.

TABLE 2 Characterization of Liposome loaded with ATR inhibitors.Drug/lipid Drug/lipid Loading Liposome size, ATR ratio before ratioafter efficiency (mean ± SD) inhibitor loading loading (%) nm Compound 6743 ± 89 550 ± 64   74 ± 0.3 119 ± 28 Compound 2 642 ± 10 639 ± 22  100± 5   130 ± 34 Compound 5 732 ± 58 619 ± 110  84 ± 8   120 ± 29 Compound3 646 ± 28 628 ± 6    97 ± 5   120 ± 27 Compound A 736 ± 57 716 ± 41  97 ± 2   121 ± 26 Compound 1 600-800 Not loadable. Formed aggregatesand precipitated 379 369 98 122 ± 24

Example 8: General Description of PK Study of Liposomal ATRi

FIG. 5 is a graph showing the blood pharmacokinetics of liposomal ATRinhibitors. Liposomal formulations of ATR inhibitors were prepared asdescribed in Example 7. The liposomes were administered intravenously ata dose of 20 mg drug/kg to three 7-9-week-old female CD-1 mice (CharlesRiver) (body weight about 25 g). Blood samples were collected intolithium heparin tubes by bleeding from saphenous vein at 0.08, 1.5, 4, 8and 24 h time points. Plasma was separated from the cell fraction bycentrifugation at 10000 rpm for 5 min. Drugs were extracted byincubation of plasma samples with 200 μl of 1% acidic acid in methanol(1% Ac/MeOH) to at least 2 hours at −80° C. Plasma proteins were spundown by centrifugation at 15000 rpm for 20 min. Then 75 μl ofsupernatant was transferred to HPLC vials (Thermo Scientific, Cat#C4011-LV1) and additional 75 μl of 1% Ac/MeOH were added. Drug contentwas analyzed by HPLC with each sample measured in duplicate. The datawere expressed as the % injected dose plotted against post injectiontime. As shown in FIG. 5 , liposomal formulation of Compound 1, Compound3 and Compound 2 were unstable in the circulation with liposomalCompound 1 and Compound 3 un-detectable by HPLC analysis at 24 hour timepoint and liposomal Compound 2 un-detectable already at 8 h time point.Two liposomal formulations Compound 6 and Compound 5 have goodcirculation longevity with above 16% of the initial injected doze after24 hours. Table 2 below summarizes the blood PK curves. LiposomalCompound 6 and Compound 5 show the highest plasma half-lives compare toother liposomal examples.

TABLE 3 Pharmacokinetics parameters of liposomal ATR inhibitors C_(max)AUC Vd Cl T_(1/2) % of ID Drug (mg/ml) (mg/ml*h) (ml) (ml/h) (h) after24 h Ls-Compound 3 0.271 1.659 2.020 0.330 4.24 0.0 Ls-Compound 6 0.3033.021 1.655 0.166 6.91 16.4 Ls-Compound 2 0.080 0.160 5.806 2.890 1.390.0 Ls-Compound 5 0.227 2.504 2.012 0.182 7.65 17.2 Ls-Compound A 0.2371.902 0.712 0.089 5.56 9.0 Ls-Compound 1 0.053 0.043 6.358 7.841 0.560.0

Example 9A: In vivo Antitumor Efficacy and Tolerability of LS-Compound APrepared Using TEA.SOS Against Cervical Cancer Xenografts in Mice

FIG. 6 is a graph showing the antitumor efficacy of liposomal Compound Ain combinations with MM-398 in cervical MS571 xenograft model, asdescribed in Example 9A.

FIG. 7 is a graph showing the antitumor efficacy of liposomal Compound Ain combinations with MM-398 in cervical C33A xenograft model, asdescribed in Example 9A.

FIG. 8 is a graph showing the antitumor efficacy of liposomal Compound Ain combinations with MM-398 in cervical C33A xenograft model, accordingto Example 9A.

FIG. 9 is a graph showing the tolerability of liposomal Compound A incombination with MM-398 in cervical MS751, according to Example 9A.

Antitumor efficacy of liposomes loaded with an ATR inhibitor Compound A(Ls-Compound A) in combinations with MM-398 (liposomal Irinotecan) wasstudied in the model of human cervical MS751, C33A and SiHa cell line.The cells were obtained from American Type Culture Collection(Rockville, MD) and propagated in RPMI medium supplemented with 10%fetal calf serum, 50 U/mL penicillin G, and 50 μg/mL of streptomycinsulfate at 37° C., 5% CO₂ as recommended by the supplier. NCR nu/nuhomozygous athymic male nude mice (4-5 week old, weight at least 16 g)were obtained from Charles River. The mice were inoculatedsubcutaneously in the right flank with 0.1 mL of the suspensioncontaining 5×106 cells suspended in PBS supplemented with 30% Matrigel.When tumors achieved the size between 150 mm³ and 350 mm³ the animalswere assigned to the treatment groups according to the following method.The animals were ranked according to the tumor size, and divided into 6categories of decreasing tumor size. Four treatment groups of 10animals/group were formed by randomly selecting one animal from eachsize category, so that in each treatment group all tumor sizes wereequally represented.

The animals received four tail vein injections, at the intervals of 7days, of the following preparations: 1) Control (HEPES-buffered salinepH 6.5); 2) MM-398 at dose 2 or 5 mg/kg per injection; 3) LiposomalCompound A at 20 or 60 mg/kg per injection; 4) MM-398 followed byinjections of liposomal Compound A with a 24 h interval. Liposomes forinjections were prepared as described in Example 7. The animal weightand tumor size were monitored twice weekly. The tumor progression wasmonitored by palpation and caliper measurements of the tumors along thelargest (length) and smallest (width) axis twice a week. The tumor sizeswere determined twice weekly from the caliper measurements using theformula (Geran, R. I., et al., 1972 Cancer Chemother. Rep. 3:1-88):

Tumor volume=[(length)×(width)²]/2

To assess treatment-related toxicity, the animals were also weightedtwice weekly. The animals were observed for 60 days following tumorinoculation. When the tumors in the group reached 10% of the mouse bodyweight, the animals in the group were euthanized. Average tumor volumesacross the groups were plotted together and compared over time. As shownin FIGS. 6, 7, and 8 , combination of liposomal ATR inhibitor Compound Awith MM-398 has a significantly stronger antitumor effect compare toMM-398 and liposomal Compound A alone in all three xenograft models. Thetreatment related toxicity was assessed by the dynamics of animals' bodyweight (FIG. 9 ). Neither group revealed any significant toxicity. Theweight of the animals in all treated groups was comparable to thecontrol group and was consistently increasing. Thus, the liposomeformulation of ATR inhibitor Compound A showed increased antitumoractivity in the studied tumor models without an appreciable increase intoxicity.

Example 9B: MM-398 Irinotecan Liposome Manufacturing

The MM398 used in Example 9A and elsewhere herein is an irinotecanliposome that can be prepared in a multi-step process. First, lipids aredissolved in heated ethanol. The lipids can include DSPC, cholesteroland MPEG-2000-DSPE combined in a 3:2:0.015 molar ratio. Preferably, theliposomes can encapsulate irinotecan sucrose octasulfate (SOS)encapsulated in a vesicle consisting of DSPC, cholesterol andMPEG-2000-DSPE combined in a 3:2:0.015 molar ratio. The resultingethanol-lipid solution is dispersed in an aqueous medium containingsubstituted amine and polyanion under conditions effective to form aproperly sized (e.g. 80-120 nm) essentially unilamellar liposomecontaining the substituted amine (in the ammonium form) and polyanionencapsulated within a vesicle formed from the dissolved lipids. Thedispersing can be performed, e.g., by mixing the ethanolic lipidsolution with the aqueous solution containing a substituted amine andpolyanion at the temperature above the lipid transition temperature,e.g., 60-70° C., and extruding the resulting hydrated lipid suspension(multilamellar liposomes) under pressure through one or moretrack-etched, e.g. polycarbonate, membrane filters with defined poresize, e.g. 50 nm, 80 nm, 100 nm, or 200 nm. The substituted amine can betriethylamine (TEA) and the polyanion can be sucrose octasulfate (SOS)combined in a stoichiometric ratio (e.g., TEA₈SOS) at a concentration ofabout 0.4-0.5N. All or substantially all non-entrapped TEA or SOS isthen removed (e.g., by gel-filtration, dialysis or ultrafiltration)prior to contacting the liposome with irinotecan under conditionseffective to allow the irinotecan to enter the liposome in exchange withTEA leaving the liposome. The conditions can include one or moreconditions selected from the group consisting of: addition of theosmotic agent (e.g., 5% dextrose) to the liposome external medium tobalance the osmolality of the entrapped TEA-SOS solution and/or preventosmotic rupture of the liposomes during the loading, adjustment and/orselection of the pH (e.g. to 6.5) to reduce the drug and/or lipiddegradation during the loading step, and increase of the temperatureabove the transition temperature of the liposome lipids (e.g., to 60-70°C.) to accelerate the transmembrane exchange of TEA and irinotecan. Theloading of irinotecan by exchange with TEA across the liposomepreferably continues until all or substantially all of the TEA isremoved from the liposome, thereby exhausting its concentration gradientacross the liposome. Preferably, the irinotecan liposome loading processcontinues until the gram-equivalent ratio of irinotecan tosucrooctasulfate is at least 0.9, at least 0.95, 0.98, 0.99 or 1.0 (orranges from about 0.9-1.0, 0.95-1.0, 0.98-1.0 or 0.99-1.0). Preferably,the irinotecan liposome loading process continues until the TEA is atleast 90%, at least 95%, at least 98%, at least 99% or more of the TEAis removed from the liposome interior. The irinotecan can formirinotecan sucrosofate within the liposome, such as irinotecan andsucrose octasulfate in a molar ratio of about 8:1. Next, any remainingextra-liposomal irinotecan and TEA is removed to obtain the irinotecanliposome using, e.g., gel (size exclusion) chromatography, dialysis, ionexchange, or ultrafiltration methods. The liposome external medium isreplaced with injectable, pharmacologically acceptable fluid, e.g.,buffered isotonic saline. Finally, the liposome composition issterilized, e.g., by 0.2-micron filtration, dispensed into dose vials,labeled and stored, e.g., upon refrigeration at 2-8° C., until use. Theliposome external medium can be replaced with pharmacologicallyacceptable fluid at the same time as the remaining extra-liposomalirinotecan and TEA is removed. The extra-liposomal pH of the compositioncan be adjusted or otherwise selected to provide a desired storagestability property (e.g., to reduce formation of the lyso-PC within theliposome during storage at 4° C. over 180 days), for example bypreparing the composition at a pH of about 6.5-8.0, or any suitable pHvalue there between (including, e.g., 7.0-8.0, and 7.25).

DSPC, cholesterol (Chol), and PEG-DSPE were weighed out in amounts thatcorresponded to a 3:2:0.015 molar ratio, respectively (e.g., 1264mg/412.5 mg/22.44 mg). The lipids were dissolved in chloroform/methanol(4/1 v/v), mixed thoroughly, and divided into 4 aliquots (A-D). Eachsample was evaporated to dryness using a rotary evaporator at 60° C.Residual chloroform was removed from the lipids by placing under vacuum(180 μton) at room temperature for 12 h. The dried lipids were dissolvedin ethanol at 60° C., and pre-warmed TEA₈SOS of appropriateconcentration was added so that the final alcohol content was 10% (v/v).The lipid concentration was 75 mM. The lipid dispersion was extruded atabout 65° C. through 2 stacked 0.1 μm polycarbonate membranes(Nucleopore) 10 times using Lipex thermobarrel extruder (NorthernLipids, Canada), to produce liposomes with a typical average diameter of95-115 nm (determined by quasielastic light scattering). The pH of theextruded liposomes was adjusted with 1 N NaOH to pH 6.5 as necessary.The liposomes were purified by a combination of ion-exchangechromatography and size-exclusion chromatography. First, DOWEX IRA 910resin was treated with 1 N NaOH, followed by 3 washes with deionizedwater and then followed by 3 washes of 3 N HCl, and then multiple washeswith water. The liposomes were passed through the prepared resin, andthe conductivity of the eluted fractions was measured by using aflow-cell conductivity meter (Pharmacia, Upsalla, Sweden). The fractionswere deemed acceptable for further purification if the conductivity wasless than 15 μS/cm. The liposome eluate was then applied to a SephadexG-75 (Pharmacia) column equilibrated with deionized water, and thecollected liposome fraction was measured for conductivity (typicallyless than 1 μS/cm). Cross-membrane isotonicity was achieved by additionof 40% dextrose solution to a final concentration of 5% (w/w) and thebuffer (Hepes) added from a stock solution (0.5 M, pH 6.5) to a finalconcentration of 10 mM.

A stock solution of irinotecan was prepared by dissolving irinotecan⋅HCltrihydrate powder in deionized water to 15 mg/mL of anhydrousirinotecan-HCl, taking into account water content and levels ofimpurities obtained from the certificate of analysis of each batch. Drugloading was initiated by adding irinotecan at 500 g/mol liposomephospholipid and heating to 60±0.1° C. for 30 min in a hot water bath.The solutions were rapidly cooled upon removal from the water bath byimmersing in ice cold water. Extraliposomal drug was removed by sizeexclusion chromatography, using Sephadex G75 columns equilibrated andeluted with Hepes buffered saline (10 mM Hepes, 145 mM NaCl, pH 6.5).The samples were analyzed for irinotecan by HPLC and phosphate by themethod of Bartlett (see Phosphate Determination).

One preferred example of a storage stable irinotecan liposome describedherein is the product that will be marketed as ONIVYDE (irinotecanliposome injection). ONIVYDE is a topoisomerase inhibitor, formulatedwith irinotecan hydrochloride trihydrate into a liposomal dispersion,for intravenous use. ONIVYDE indicated for the treatment of metastaticadenocarcinoma of the pancreas after disease progression followinggemcitabine-based therapy.

ONIVYDE is a storage stabilized liposome having a pH of about 7.25. TheONIVYDE product contains irinotecan sucrosofate encapsulated in aliposome, obtained from an irinotecan hydrochloride trihydrate startingmaterial. The chemical name of irinotecan is(S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3′,4′:6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4′bipiperidine]-1′-carboxylate.The dosage of ONIVYDE can be calculated based on the equivalent amountof irinotecan trihydrate hydrochloride starting material used to preparethe irinotecan liposomes, or based on the amount of irinotecan in theliposome. There are about 866 mg of irinotecan per gram of irinotecantrihydrate hydrochloride. For example, an ONIVYDE dose of 80 mg based onthe amount of irinotecan hydrochloride trihydrate starting materialactually contains about 0.866×(80 mg) of irinotecan in the final product(i.e., a dose of 80 mg/m² of ONIVYDE based on the weight of irinotecanhydrochloride starting material is equivalent to about 70 mg/m² ofirinotecan in the final product). ONIVYDE is a sterile, white toslightly yellow opaque isotonic liposomal dispersion. Each 10 mLsingle-dose vial contains 43 mg irinotecan free base at a concentrationof 4.3 mg/mL. The liposome is a unilamellar lipid bilayer vesicle,approximately 110 nm in diameter, which encapsulates an aqueous spacecontaining irinotecan in a gelated or precipitated state as the sucroseoctasulfate salt. The vesicle is composed of1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 6.81 mg/mL,cholesterol 2.22 mg/mL, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidyl ethanolamine (MPEG-2000-DSPE) 0.12 mg/mL.Each mL also contains 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) as a buffer 4.05 mg/mL andsodium chloride as an isotonicity reagent 8.42 mg/mL. Each vial ofONIVYDE contains 43 mg/10 mL irinotecan free base as a white to slightlyyellow, opaque, liposomal dispersion in a single-dose vial.

In one example of the invention, an ONIVYDE unit dosage form is apharmaceutical composition comprising an amount of irinotecanencapsulated in a liposome that provides a total amount of about 70 mg/m2 of irinotecan, providing an amount of irinotecan equivalent to 80mg/m² irinotecan hydrochloride trihydrate, and less than about 20%lyso-PC. The unit dosage form can be an intravenous formulation having atotal volume of about 500 mL. ONIVYDE is prepared for administering bydiluting the isotonic liposomal dispersion from the vial as follows:withdraw the calculated volume of ONIVYDE from the vial. ONIVYDE isdiluted in 500 mL 5% Dextrose Injection, USP or 0.9% Sodium ChlorideInjection, USP and mix diluted solution by gentle inversion; protectdiluted solution from light and administer diluted solution within 4hours of preparation when stored at room temperature or within 24 hoursof preparation when stored under refrigerated conditions [2° C. to 8° C.(36° F. to 46° F.)].

ONIVYDE (irinotecan liposome injection) is indicated, in combinationwith 5-fluorouracil and leucovorin, for the treatment of patients withmetastatic adenocarcinoma of the pancreas that has progressed followinggemcitabine-based therapy. Administer ONIVYDE prior to leucovorin andfluorouracil. The recommended dose of ONIVYDE is 70 mg/m2 irinotecanadministered by intravenous infusion over 90 minutes every 2 weeks. Therecommended starting dose of ONIVYDE in patients known to be homozygousfor the UGT1A1*28 allele is 50 mg/m² irinotecan administered byintravenous infusion over 90 minutes. The dose of ONIVYDE can beincreased to 70 mg/m² as tolerated in subsequent cycles. There is norecommended dose of ONIVYDE for patients with serum bilirubin above theupper limit of normal. ONIVYDE is infused as a diluted solutionintravenously over 90 minutes.

Suitable treatment regimens include ONIVYDE 70 mg/m² with (l+d racemicform) leucovorin 400 mg/m² (or 200 mg/m² of the active 1 form ofleucovorin) and fluorouracil 2,400 mg/m² over 46 hours every 2 weeks(ONIVYDE/5-FU/LV; n=117), ONIVYDE 100 mg/m² every 3 weeks (n=147), orleucovorin 200 mg/m² and fluorouracil 2000 mg/m² over 24 hours weeklyfor 4 weeks followed by 2 week rest (5-FU/LV; n=134).

Example 10: In Vivo Antitumor Efficacy and Tolerability of Ls-Compound 5Prepared Using TEA.SOS Against Lung Cancer Xenografts in Mice

FIG. 10A and FIG. 10B are each a graph, showing the efficacy ofliposomal Compound 5 in combination with MM-398 in NCI-H2170 (FIG. 10A)or DMS-114 (FIG. 10B) mice xenograft models, as discussed in Example 10.

FIG. 11A and FIG. 11B are each a graph showing the Kaplan-Meyer survivalcurves representing efficacy of liposomal Compound 5 in combination withMM-398 in NCI-H2170 (FIG. 11A) and DMS-114 (FIG. 11B) a mouse xenograftmodel, as discussed in Example 10.

FIG. 12A and FIG. 12B are each a graph showing Tolerability of liposomalCompound 5 in combination with MM-398 in NCI-H2170 (FIG. 12A) or DMS-114(FIG. 12B) in a mouse xenograft model.

Antitumor efficacy of liposomes loaded with an ATR inhibitor Compound 5in combinations with MM-398 (liposomal Irinotecan) and was studied inthe model of human NCI-H2170 (lung squamous cell carcinoma) and DMS-114(small cells lung carcinoma) lung cell lines.

The cells were obtained from American Type Culture Collection(Rockville, MD) and propagated in RPMI medium supplemented with 10%fetal calf serum, 50 U/mL penicillin G, and 50 μg/mL of streptomycinsulfate at 37° C., 5% CO₂ as recommended by the supplier. NCR nu/nuhomozygous athymic male nude mice (4-5 week old, weight at least 16 g)were obtained from Charles River. The mice were inoculatedsubcutaneously in the right flank with 0.1 mL of the suspensioncontaining 5×10⁶ cells suspended in PBS supplemented with 30% Matrigel.When tumors achieved the size between 150 mm³ and 350 mm³ the animalswere assigned to the treatment groups according to the following method.The animals were ranked according to the tumor size, and divided into 6categories of decreasing tumor size. Four treatment groups of 10animals/group were formed by randomly selecting one animal from eachsize category, so that in each treatment group all tumor sizes wereequally represented. The animals received four tail vein injections, atthe intervals of 7 days, of the following preparations: 1) Control(HEPES-buffered saline pH 6.5); 2) MM-398 at dose 5 mg/kg per injection;3) Liposomal Compound 5 at 80 mg/kg per injection; 4) MM-398 followed byinjections of liposomal Compound 5 with a 24 h interval. Liposomes forinjections were prepared as described in Example 7. MM-398 is describedin Example 9B.

The animal weight and tumor size were monitored twice weekly. The tumorprogression was monitored by palpation and caliper measurements of thetumors along the largest (length) and smallest (width) axis twice aweek. The tumor sizes were determined twice weekly from the calipermeasurements using the formula:

Tumor volume=[(length)×(width)²]/2

To assess treatment-related toxicity, the animals were also weightedtwice weekly. When the tumors in the group reached 10% of the mouse bodyweight, the animals in the group were euthanized. Average tumor volumesacross the groups were plotted together and compared over time.

As demonstrated in FIGS. 10A and 10B and FIGS. 11A and 11B, liposomalATR inhibitor Compound 5 significantly improved antitumor efficacy ofMM-398 in both lung xenograft models. The combinational treatment ofliposomal ATR inhibitor and MM-398 did not affect the animals' bodyweight (FIGS. 12A and 12B).

Example 11: Comparison of In Vivo Antitumor Efficacy of LiposomalInhibitors Ls-Compound 5 and LS-Compound A in Combination with MM-398Against Lung Cancer Xenografts in Mice

FIGS. 13A and 13B are graphs showing the efficacy of liposomal Compound5 in combination with MM-398 in Calu-6 (FIG. 13A) or COLO-699 (FIG. 13B)mice xenograft models. For example, the data in FIGS. 13A and 13B showthat while liposomal ATR inhibitor Compound 5 significantly improved theantitumor efficacy of MM-398 in both models, Compound A formulated inliposomes was active only in COLO-699 xenograft model.

Antitumor efficacy of liposomes loaded with an ATR inhibitor Compound 5in combinations with MM-398 (liposomal Irinotecan) was compared withliposomal formulation of Compound A in the xenograft model of humanCalu-6 and COLO-699 lung cell lines.

The cells were obtained from American Type Culture Collection(Rockville, MD) and propagated in RPMI medium supplemented with 10%fetal calf serum, 50 U/mL penicillin G, and 50 μg/mL of streptomycinsulfate at 37° C., 5% CO₂ as recommended by the supplier. NCR nu/nuhomozygous athymic male nude mice (4-5 week old, weight at least 16 g)were obtained from Charles River. The mice were inoculatedsubcutaneously in the right flank with 0.1 mL of the suspensioncontaining 5×106 cells suspended in PBS supplemented with 30% Matrigel.When tumors achieved the size between 150 mm³ and 350 mm³ the animalswere assigned to the treatment groups according to the following method.The animals were ranked according to the tumor size, and divided into 6categories of decreasing tumor size. Four treatment groups of 10animals/group were formed by randomly selecting one animal from eachsize category, so that in each treatment group all tumor sizes wereequally represented. The animals received four tail vein injections, atthe intervals of 7 days, of the following preparations: 1) Control(HEPES-buffered saline pH 6.5); 2) MM-398 at dose 10 or 20 mg/kg perinjection; 3) Liposomal Compound 5 at 80 mg/kg per injection; 4)Liposomal Compound A at 80 mg/kg per injection; 5) MM-398 followed byinjections of liposomal Compound 5 with a 24 h interval. 6) MM-398followed by injections of liposomal Compound A with a 24 h interval.Liposomes for injections were prepared as described in Example 7.

The animal weight and tumor size were monitored twice weekly. The tumorprogression was monitored by palpation and caliper measurements of thetumors along the largest (length) and smallest (width) axis twice aweek. The tumor sizes were determined twice weekly from the calipermeasurements using the formula:

Tumor volume=[(length)×(width)²]/2

To assess treatment-related toxicity, the animals were also weightedtwice weekly. When the tumors in the group reached 10% of the mouse bodyweight, the animals in the group were euthanized. Average tumor volumesacross the groups were plotted together and compared over time.

Example 12: Free Drug Combination Screening Study

FIG. 14A. is a graph showing the in vitro monotherapy cell kill ofCompound 6 and Compound 5 in a panel of lung cancer cell lines. Compound5 is more efficacious with IC₅₀ ˜3 (=100.5) folds lower than Compound 6.FIG. 14B is a graph showing the effect of Compound 5 vs. Compound 6 incombination with three chemotherapeutic agents (Carboplatin,Gemcitabine, Compound B). The figure compares the IC₅₀ in log (μM) ofthe combination of the chemotherapeutic agents with 1 μg/ml of Compound6 or Compound 5. The addition of Compound 5 to the chemotherapeuticagents was more potent (lower IC₅₀) than Compound 6 in all testedcytotoxic agents and in all cell lines except one.

FIG. 15 is a graph showing the combination (Example 2+Compound B) IC₅₀shift in Sum190PT cell line (TNBC).

FIG. 16A and FIG. 16B are graphs showing a comparison of IC50 shifts ofthe compound of Example 2 with Compound B vs combination of Compound Awith Compound B in MDA-MB-453 cell line (TNBC).

Culture/Treatment Condition

In vitro efficacy study was done using CELLTITER-GLO Luminescent CellViability Assay (Promega) with Corning Cat #3707 384 well White Clearbottom plates. Cells were plated (1000 cells/well) in 384 well formatand allowed to incubate at 37° C. for 24 hours. Monotherapy drugs wereadded at the 24 hr time point and then allowed to incubate at 37° C. for24 hours. At the 48 hr time point the drugs in media were removed,washed with PBS, and fresh media was added. Cells were then allowed toincubate at 37° C. for 72 hours. For combination studies, cells wereexposed to Carboplatin or Compound B or Gemcitabine for 24 hours, thenthe chemotherapeutic agent was removed and cells were exposed to thesecondary compound (ATR inhibitor) for 24 hours. Cells were cultured infresh media for an additional 48 hours.

At the 120 hr time point media was removed and CELLTITER-GLO (CTG)reagent was added (1:1 ratio with PBS). Plates were read using aluminometer (Envision Multilabel reader).

Data Analysis:

Data was analyzed using an in-house algorithm developed using Matlab(Mathworks, Natick MA). In summary, average CTG mean luminescent valueswere computed for 4 replicate wells. Outlier detection was performed bycomputing the coefficient of variation (CV>20%) and outliers wereremoved from the average. CTG values were normalized based on a controlnon-treated well. Drug concentration in microMolar (μM) was logtransformed prior to fitting to a 4 parameter logistic curve.

$y = {b + \frac{( {a - b} )}{( {1 + {10^{{({{{IC}50} - C})}*{slope}}}} )}}$

-   -   Where C: concentration of drug, y: normalized CTG value, a: top        asymptote (represents maximum cell kill), b: bottom asymptote        (constrained 0.8-1.2), IC50, slope: logistic curve slope.

Data quality control was performed to ensure that the concentrationrange is optimal according to these rules: (1) if the lowestconcentration kills more than 70% of the cells the concentration rangeis deemed too potent (2) if the highest concentration kills less than30% of the cells, the concentration range is deemed low or the cell lineis too resistant. Additionally, goodness of the fit was evaluated usingR² and R²<0.9 is flagged as a bad fit.

Statistical analysis was performed using JMP (SAS Institute Inc., NC)and p<0.05 was considered significant.

Example 13: ATR Activity Determination

An EC₅₀ determination was conducted for the supplied set of compoundsagainst the kinase ATR/ATRIP(h), using a linear enzyme concentration inthe Eurofins ATR/ATRIP HTRF assay using GST tagged full length p53 asthe substrate.

The activity of ATR/ATRIP(h) at an ATP concentration within 15 μM of theKm was determined at 9 concentrations of compound with semi-logdilutions from 10 μM. ATR/ATRIP phosphorylation of p53 on Ser15 wasmeasured via formation of an energy transfer complex consisting of aEuropium-labelled anti-phospho Ser15 p53 antibody and an anti-GST-d2antibody. All data points were performed in duplicate with DMSO controlsand EDTA blanks.

ATR/ATRIP(h) was pre-diluted in 25 mM HEPES pH 8.0, 0.01% Brij-35, 1%Glycerol, 5 mM DTT, 1 mg/mL BSA and assayed in 25 mM HEPES pH 8.0, 0.01%Brij-35, 1% Glycerol, 10 mM MnCl₂ using 30 nM GST,cMyc p53-(hu,FL) asthe substrate. The reaction was initiated with the addition of ATP to afinal concentration of 10 μM.

The individual replicates were expressed in terms of the % of the DMSOpositive control activity. The mean activity (% control) of eachinhibitor concentration was plotted against the inhibitor concentration,and EC₅₀ values were determined using GRAPHPAD PRISM.

The data expressed as % control activity were plotted against inhibitorconcentration, and fitted to a four parameter logistic using GRAPHPADPRISM. Graphs for each compound are shown alongside the data in theaccompanying Excel report. A summary of the compound potencies is shownbelow.

TABLE 4 Compound ID Kinase ATP μM EC₅₀* nM Top Bottom Hill r² Compound 6ATR ATRIP(h) 10 470.8 101 −2 −0.768 0.998 Compound 2 ATR ATRIP(h) 10139.8 94 1 −0.911 0.998 Compound 5 ATR ATRIP(h) 10 233.9 95 1 −0.8190.996 Compound 1 ATR ATRIP(h) 10 49.6 95 −1 −0.772 0.994 Compound 4 ATRATRIP(h) 10 353.4 97 1 −0.860 0.997 Compound 3 ATR ATRIP(h) 10 196.1 98−3 −0.776 0.999 Compound A ATR ATRIP(h) 10 <1 ND ND ND ND

Example 14: ATM Activity Determination

Estimated IC₅₀ values are as follows (obtained using STANDARDKINASEPROFILER):

TABLE 5 Compound Kinase IC₅₀ (nM) Compound 6 ATM(h) >10,000 Compound 2ATM(h) 4817 Compound 5 ATM(h) >10,000 Compound 1 ATM(h) 3864 Compound 4ATM(h) 6869 Compound 3 ATM(h) >10,000 Compound A ATM(h) 42

Example 15: Detecting Total and Phospho-ATR with Western Blot Analysis

Referring to FIG. 17 : Lung cancer cells DMS-114 were exposed either toGemcitabine [16 nM] or ATR inhibitor (Compound A or Compound 5 [1 μM])alone or in combination in vitro. Whole cell protein lysates weregenerated after 1, 3, 6 and 24 hours using a 2% SDS containing celllysis buffer and stored at −80° C. until all samples were collected andrun together at the same time for the WesternBlot analysis. Thefollowing proteins and phosphoproteins of interest were detected byantibodies purchased from Cell Signaling Technology (see methods andmaterials part below): total and phospho-ATR (S428), phospho-CHK1 (S317and S345), yH2AX, beta-actin.

Results in FIG. 17 : Total and phospho-ATR signal is strongly reducedover time with the addition of either of the two ATR inhibitors(Compound A and Compound 5) and not in control or Gemcitabine alonetreatment (compare e.g. lane 1, 2 with 3, 4 or 5, 6 at 24 hrs). Inresponse to Gemcitabine, the phospho-CHK1 (S317 and S345) signaling issignificantly increased over the period of 24 hrs. The addition of anyof the used ATR inhibitors (Compound A or Compound 5) abrogates thephosphorylation signal of CHK1. The reduction of the ATR protein anddownstream CHK1 signaling by both inhibitors confirms the specificon-target effects of both molecules. More importantly, our hypothesis isthat the loss of the cell cycle check-point, driven by the CHK1phosphorylation signaling that supposed to lead to cell cycle arrest andrepair of DNA damage, induces cell death by accumulation of DNA damage.The increased yH2AX signal can be seen as a surrogate for themeasurement of increased toxicity and accumulation of DNA damage.

Cell Culture

U2OS and all other cells were grown in (RPMI) supplemented with 10%fetal bovine serum (FBS) and antibiotics. Imaging and time-lapsecompatible NucLight red cell lines were generated as recommend by EssenBioscience Inc. using lentiviral particles, infection and puromycinselection protocols.

Antibodies and Western-Blots

All target and phospho-specific antibodies were purchased from CellSignaling and/or Epitomics and were used at 1:1000 dilutions. A list ofall antibodies is available in Table 6. Standard antibody based Westernblot and immunohistochemistry protocols were used. Briefly, 2% SDS basedcell lysis buffer was used to collect whole cell lysate for WesternBlotting. Secondary antibodies and staining protocols were purchased andfollowed regarding LiCor Biosciences and/or BD Bioscience.

TABLE 6 Target Company Catalog # ATR Cell Signaling  2790 Technology ATRphospho Cell Signaling  2853 (S428) Technology CHK1 phospho CellSignaling 12302 (S317) Technology CHK1 phospho Cell Signaling  2348(S345) Technology H2AX phospho Cell Signaling  9718 (S139) Technologybeta-actin Cell Signaling  4970 Technology

Whole Cell Lysis Protocol

Cells were grown and treated in a 6 cm dish scale. To harvest cells,medium was removed and quickly replaced by ice cold PBS. PBS was thenreplaced with 250 μL of 2% SDS lysis buffer. After five minutes ofincubation, the lysed cells were scraped off and loaded onto a celllysate homogenizer microcentrifuge spin-column (QIAshredder, Qiagen,Cat.-Nr. 79656). The filtrate was then loaded onto a 0.2 μm centrifugalfilter column (Nanosep MF 0.2_m, Pall, Cat.-Nr. ODM02C34). The lysateswere then stored at −80° C.

2% SDS containing lysis buffer (Table 7) was modified according toSteven et al., “Protein microarrays for multiplex analysis of signaltransduction pathways,” Nat Med 10, no. 12 (December 2004): 1390-1396.

TABLE 7 Titrated pH = 6.8 Final Conc. Trizma-Base 50 mM SDS 2% Glycerol5% EDTA  5 mM NaF  1 mM

Example 16: Broad Kinase Panel Screening (359)

Major hits out of 359 were: ALK, ARK, c-MER, CLK1, DYRK, GSK3a, GSK3b,FLT2, FLT3, MLK1, SIK2, TNIK, and YSK4.

TABLE 8 Compound Log Kinase: 5 (M) IC₅₀ Kinase: nM ALK1/ACVRL1 3.57E−08−7.45 ALK1/ACVRL1 35.7 ALK2/ACVR1 1.66E−08 −7.78 ALK2/ACVR1 16.6ARK5/NUAK1 1.68E−08 −7.78 ARK5/NUAK1 16.8 CLK1 1.34E−07 −6.87 CLK1 134CLK4 8.86E−08 −7.05 CLK4 88.6 DDR1 1.91E−07 −6.72 DDR1 191 DYRK27.18E−08 −7.14 DYRK2 71.8 FLT4/VEGFR3 6.34E−08 −7.20 FLT4A/VEGFR3 63.4GSK3a 5.73E−09 −8.24 GSK3a 5.73 GSK3b 6.30E−08 −7.20 GSK3b 63MLK1/MAP3K9 1.13E−08 −7.95 MLK1/MAP3K9 11.3 MLK2/MAP3K10 9.58E−09 −8.02MLK2/MAP3K10 9.58 MLK3/MAP3K11 5.90E−09 −8.23 MLK3/MAP3K11 5.9 PIM17.26E−08 −7.14 PIM1 72.6 PKCmu/PRKD1 1.34E−07 −6.87 PKCmu/PRKD1 134PKD2/PRKD2 1.57E−07 −6.80 PKD2/PRKD2 157 RET 7.74E−08 −7.11 RET 77.4SIK2 1.49E−07 −6.83 SIK2 149 TNIK 1.10E−07 −6.96 TNIK 110 YSK4/MAP3K192.92E−08 −7.53 YSK4/MAP3K19 29.2

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features set forth herein.

Example 17: Data Using Compound A as a Comparator

A cell death and growth assay was performed combining Compound A andGemcitabine at various concentrations in U2OS cells. The results werecompared back to a prediction of cell growth and death with thecombination of the two compounds (FIG. 18A). The number of living andapoptotic cells were also determined at set concentrations of Compound A(1 μM) and Gemcitabine (0.04 μM) in USO2 cells (FIG. 18B). The resultsshow that the combination of Compound A and Gemcitabine are better atpromoting cell death than either compound alone.

Compound A was also tested alone and in combination with SN38 at setconcentrations (1 μM and 0.2 μM, respectively) in several lung cancercell lines (NCI-H520 and NCI-H596) and U2OS cells, with the number ofcells monitored over time. The results indicate that the combination ismore effective at maintaining or reducing cell numbers over time thaneither Compound A or SN38 in isolation (FIG. 19 ).

In vitro growth assays were also performed with Compound A and SN38 incombination and alone in the cervical cancer cell line MS-751. The invitro assay shows a reduction in cell number over time with thecombination as compared to either compound alone (FIG. 20 ).

Example 18: Comparison of Compound A and Compound 5 in Combination withGemcitabine or SN38

In vitro cell death and growth assays were performed combining CompoundA or Compound 5 with Gemcitabine at various concentrations in U2OS,H358, and A549 cell lines. The results indicate the concentrations ofeach compound and Gemcitabine that are required to trigger cell death(FIG. 21A). Set concentrations of Compound 5 and Gemcitabine or CompoundA and Gemcitabine were tested in USO2 and H358 cells as well. In allcases, relative cell proliferation is reduced with the combinationscompared to the drugs alone (FIG. 21B-C).

Compound A or Compound 5 was used in combination with SN38 in A549cells. Cell growth was measured over time and at a range ofconcentrations. The growth curve at the optimal Compound/SN38concentration is highlighted (FIG. 22A). Relative proliferation of A549cells was measured with either Compound A or Compound 5 alone at a rangeof concentrations (FIG. 22B).

The IC50 values were determined in several cell lines using a setconcentration of Gemcitabine with varying concentrations of Compound Aor Compound 5 (FIG. 23 ).

A summary of the lung cancer cell lines that are responsive to CompoundA or Compound 5 in combination with Gemcitabine or SN38 is provide inFIG. 24 .

Example 19: On-Target and Off-Target Comparisons of Compound A andCompound 5

Various ATR inhibitors were tested for their ability to inhibit ATR(on-target) and ATM (off-target). Inhibition is reported as IC50 in nM(FIG. 25A). Additional “off-target” kinases were tested with Compound Aor Compound 5 as well (FIG. 25B).

On-target testing was performed with Compound A or Compound 5 in A549lung cancer cells (FIG. 26A-B), H23 lung cancer cells (FIG. 26C-D), andDMS-114 cells (FIG. 26E-F). CHK1 S345 phosphorylation, a readout of ATRinhibition, was measured by western blot. Each compound was used with afixed concentration of Gemcitabine as well.

Further on-target studies were performed on the HCC-70 TNBC, MDA-MB-468TNBC, and DMS-114 cell line. A set concentration of SN38 was used with arange of Compound A or Compound 5 concentrations. Various on-targetactivity parameters were tested and measured by western blot (FIG.27A-B)

Cell cycle stage profiles of SUM149 cells were determined 24 hours afteraddition of SN38 and Compound A or Compound 5 (FIG. 28 ). A largerpercentage of cells are arrested in G2 phase when receiving thecombination of drugs as compared to controls.

Example 20: Comparison of Liposomal Formulations of Compound A andCompound 5 Combined with MM398

DMS-114 lung xenograft models were used to measure the effects ofLs-Compound A or Ls-Compound 5 in combination with MM398. A set dosageof MM398 was used (5 mpk) with two different dosages of Compound A orCompound 5 (20 mpk or 80 mpk). The effects of treatment were assayed bymeasuring the levels of CHK1 S345 phosphorylation (FIG. 29 ).

The effects of the Ls-Compound A with MM398 was also tested in theSUM-149 cell line. The effects of the treatment were assayed bymeasuring phosphorylated levels of RPA2, DNAPK, CHK1, and γH2AX.Ls-Compound 5 was also tested without the combination of MM398 (FIG.30A-C).

Example 21: In Vivo Antitumor Efficacy and Tolerability of Ls-Compound 5Prepared Using TEA.SOS Against Triple Negative Breast Cancer Xenograftsin Mice

Antitumor efficacy of liposomes loaded with an ATR inhibitor Compound 5in combinations with MM-398 (liposomal Irinotecan) and was studied inthe model of human SUM-149 (triple negative breast cancer) cell line.

The cells were obtained from American Type Culture Collection(Rockville, MD) and propagated in RPMI medium supplemented with 10%fetal calf serum, 50 U/ml penicillin G, and 50 μg/mL of streptomycinsulfate at 37° C., 5% CO2 as recommended by the supplier. NCR nu/nuhomozygous athymic male nude mice (4-5 week old, weight at least 16 g)were obtained from Charles River. The mice were inoculatedsubcutaneously in the right flank with 0.1 mL of the suspensioncontaining 107 cells suspended in PBS supplemented with 30% Matrigel.When tumors achieved the size between 150 mm3 and 350 mm3 the animalswere assigned to the treatment groups according to the following method.The animals were ranked according to the tumor size, and divided into 6categories of decreasing tumor size. Four treatment groups of 10animals/group were formed by randomly selecting one animal from eachsize category, so that in each treatment group all tumor sizes wereequally represented. The animals received five tail vein injections, atthe intervals of 7 days, of the following preparations: 1) Control(HEPES-buffered saline pH 6.5); 2) MM-398 at dose 5 mg/kg per injection;3) Liposomal Compound 5 at 80 mg/kg per injection; 4) MM-398 followed byinjections of liposomal Compound 5 with a 24 h interval; 5)) MM-398followed by injections of free un-capsulated ATR inhibitor Compound A.Liposomes for injections were prepared as described in Example 10.

The animal weight and tumor size were monitored twice weekly. The tumorprogression was monitored by palpation and caliper measurements of thetumors along the largest (length) and smallest (width) axis twice aweek. The tumor sizes were determined twice weekly from the calipermeasurements using the formula:

Tumor volume=[(length)×(width)²]/2

To assess treatment-related toxicity, the animals were also weightedtwice weekly. When the tumors in the group reached 10% of the mouse bodyweight, the animals in the group were euthanized. Average tumor volumesacross the groups were plotted together and compared over time.

As demonstrated in FIG. 31 , liposomal ATR inhibitor Compound 5significantly improved antitumor efficacy of MM-398 in the xenograftmodel. The combinational treatment of liposomal ATR inhibitor and MM-398did not affect the animals' body weight (FIG. 32 ).

Example 22: Cell Line Profiling and Comparison with Incucyte Data

A panel of fluorescently labeled cell lines was profiled for variousproteins involved in the DNA damage pathway. Parental cancer cell lineswere transduced with a NucLight Red lentivirus and selected withpuromycin. Basal protein levels were measured and quantified by Westernblot analysis. When quantification was performed, the PD marker signalwas normalized to the beta actin signal. Protein levels were correlatedwith the cell line's “integral score”, a measure of the in vitrocellular response to Compound 5 and chemotherapy combination treatment.The integral score can be calculated as follows:

Referring to FIGS. 33 and 34 , cell lines were seeded in 96 well platesand each well was exposed to a dose matrix of Compound 5 andchemotherapy for 4 days. Dynamic cell viability was measured using anIncucyte assay. For each drug combination in the dose matrix, a dosecombination-specific integral score was calculated by summing the areaunder normalized cell proliferation curve. A cell line specific integralscore was computed by averaging the four largest scores over the dosematrix.

Referring to FIGS. 35 and 36 , it was observed that in lung cancer celllines exposed to Compound 5 and SN38, basal MRE11 protein levels andbasal ATM levels both correlated significantly with the cell lineintegral scores. In lung cancer cells that have a p53 compromisedbackground and were exposed to Compound 5 and SN38, the integral scorecorrelates with NBS, MRE11 and RAD50 protein levels. “Compromised p53”cells are those that have non-zero basal p53 protein levels (in mostcells, p53 levels are only visible in Western blots after treatment).(See FIG. 37 , FIG. 38 and FIG. 39 ).

Example 23: Signaling Experiment: Compound 5 vs. Compound A inCombination with SN38: 6 Different PD Markers

Referring to FIGS. 40-44 : Pharmacodynamics markers pChk1, pRPA2, pATR,pDNAPK, pChk2 and γH2AX were quantified by Western blot after cancercell lines were exposed to various doses of Compound 5 or Compound A incombination with SN38. HCC70, DMS114, MDAMB468, NCIH1299 and NCIH460cells were seeded in 12 well plates, allowed to incubate overnight thenexposed to SN38 and/or the ATR inhibitor. After 24 hours of drugexposure, cells were lysed for Western blotting. When quantification wasperformed, the PD marker signal was normalized to the beta actin signal.

Example 24: Signaling Experiment: Compound 5 vs. Compound A inCombination with Gemcitabine: 3 Different PD Markers

Referring to FIGS. 45-50 : Six fluorescently labeled cell lines wereprofiled for various proteins involved in the DNA damage pathway afterexposure to an ATR inhibitor and/or gemcitabine. Parental cancer celllines were transduced with a NucLight Red lentivirus and selected withpuromycin. Pharmacodynamics markers pChk1, pATR, and γH2AX werequantified by Western blot after cancer cell lines were exposed tovarious doses of Compound 5 or Compound A in combination with 16 nMgemcitabine. A549, NCIH23, DM5114, U2O5, HCC827 and NCIH460 cells wereseeded in 12 well plates, allowed to incubate overnight then exposed toSN38 and/or the ATR inhibitor. After 6 or 18 hours of drug exposure,cells were lysed for Western blotting. When quantification wasperformed, the PD marker signal was normalized to the beta actin signal.

1.-15. (canceled)
 16. A pharmaceutical composition comprising a compoundof formula (Ia), or a pharmaceutically acceptable salt thereof, wherein:

R′ is NR^(a)R^(b), wherein R^(a) and R^(b) are each independentlyC₁-C₄alkyl; and a pharmaceutically acceptable medium.
 17. Apharmaceutical composition comprising a compound of the formula ofCompound 5, or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable medium.


18. A pharmaceutical composition comprising a compound of the formula ofCompound 6, or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable medium.